UV Resonance Raman Structural Characterization of an (In)soluble

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UV Resonance Raman Structural Characterization of an (In)soluble Polyglutamine Peptide Ryan S Jakubek, Stephen White, and Sanford A. Asher J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b10783 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 10, 2019

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The Journal of Physical Chemistry

UV Resonance Raman Structural Characterization of an (In)soluble Polyglutamine Peptide Ryan S. Jakubek†, Stephen E. White†,§, and Sanford A. Asher†*

†

Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States

§

Molecular Biophysics and Structural Biology Program, University of Pittsburgh, Pittsburgh Pennsylvania 15260, United States

Email: [email protected]

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Abstract Fibrillization of polyglutamine (polyQ) tracts in proteins is implicated in at least ten neurodegenerative diseases. This generates great interest in the structure and the aggregation mechanism(s) of polyQ peptides. The fibrillization of polyQ is thought to result from the peptide’s insolubility in aqueous solutions; longer polyQ tracts show decreased aqueous solution solubility, which is thought to lead to faster fibrillization kinetics. However, few studies have characterized the structure(s) of polyQ peptides with low solubility. In the work here we use UV resonance Raman spectroscopy to examine the secondary structures, backbone hydrogen bonding, and side chain hydrogen bonding for a variety of solution state, solid, and fibril forms of D2Q20K2 (Q20). Q20 is insoluble in water and has a β-strand-like conformation with extensive inter- and intra-peptide hydrogen bonding in both dry and aqueous environments. We find that Q20 has weaker backbone-backbone and backbone-side chain hydrogen bonding and is less ordered compared to that of polyQ fibrils. Interestingly, we find that the insoluble Q20 will form fibrils when incubated in water at room temperature for ~5 hours. Also, Q20 can be prepared using a well-known disaggregation procedure to produce a water soluble PPII-like conformation with negligible inter- and intrapeptide hydrogen bonding and a resistance to aggregation.


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Introduction Expanded polyglutamine (polyQ) tracts in proteins and peptides induce aggregation and fibrillization.1 This polyQ-induced fibrillization is associated with at least ten neurodegenerative diseases, including Huntington’s disease (HD) and multiple spinocerebellar ataxias (SCAs).1 The mechanism of toxicity and the identity of the toxic species are still debated.2–4 The common factor for polyQ-associated neurodegenerative diseases is the presence of an expanded polyQ tract.1 In polyQ repeat diseases, longer polyQ tracts are correlated with an earlier disease symptom age-of-onset.5 Disease symptoms are only observed when the protein polyQ tract length surpasses a critical length (~≥36Q for the huntingtin protein in HD).1 This polyQ tract length dependence of disease age-of-onset is thought to result from a length-induced increase in the polyQ aggregation kinetics.6–8 This possibility was strengthened when Chen et al. showed that polyQ peptides with longer polyQ tracts have faster aggregation kinetics.6 Also, Chen et al. used aggregation rates calculated from polyQ peptides at high concentrations (~5-50 μM) to extrapolate aggregation rates for polyQ peptides at physiological concentrations (~0.1 nM).6

In these calculations, Q47 at

physiological concentrations was calculated to aggregate in ~31 years. This aggregation rate is quite similar to the HD age-of-onset (30-40 years) for patients with a Q47 tract length in the huntingtin protein. Also, Q36 and Q28 lengths were calculated to begin aggregating in 141 and 1273 years, respectively, at physiological concentrations. These putative ages of disease onset roughly agree with the age of onset for polyQ tracts with >1 week. However, incubation of DQ20 at 37°C and pH=~+7 results in fibril formation in ~1 week (Figure 1j). The UVRR spectra of DQ20 fibrils are similar to those previously observed for DQ10 fibrils (Figure 5a).17 The AmIII3S and AmI bands of DQ10 and DQ20 fibrils are similar, indicating that their secondary structures and hydrogen bonding interactions are similar (Figure S4). The UVRR spectra of DQ20 fibrils show AmIII3S bands at ~1230 cm-1 and ~1210 cm-1 that derive from Ψ angle distributions centered at ~145° and ~123° (Figure 6a). As discussed previously by Punihaole et al., Ψ angle distributions centered at ~145° are characteristic of antiparallel β-sheet structures, while those centered at ~123° are characteristic of parallel β-sheet structure.


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Figure 5: (a) 204 nm, (b) 197-204 nm, and (c) 204-(197-204) nm UVRR spectra of DQ20 fibrils.



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Figure 6: (a) Ψ angle distribution and (b) TEM image of DQ20 fibrils.

In the 197-204 nm difference spectrum, the AmIP band maximum of DQ20 fibrils occurs at ~1662 cm-1 (Figure 5b). As discussed above, this is characteristic of Gln side chain C=O groups involved in strong side chain-side chain and/or side chain-backbone hydrogen bonding. Using eq. 1 we estimate that the ΔHint for Gln side chain C=O groups in DQ20 fibrils is ~-5.7 kcal/mol, similar to that previously found for DQ10 fibrils.30 Similarly, an AmIS band is observed in the 204-(197-204) nm difference spectrum (Figure 5c) of DQ20 fibrils at ~1663 cm-1 that is characteristic of backbone amides involved in β-sheet backbone-backbone hydrogen bonding as previously observed for Q10 fibrils.17,30 Overall, the AmIP and AmIS band frequencies show that 24 ACS Paragon Plus Environment

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both backbone and side chain amide C=O groups in DQ20 fibrils are involved in strong peptidepeptide hydrogen bonding.

Solubility of NDQ20 As discussed above, NDQ20 does not form clear solutions in water (Figure 1d). We used UV absorbance to detect the presence of any soluble peptide in the supernatant after ultracentrifugation of NDQ20. We found that the NDQ20 supernatant contains significant absorbance suggesting the presence of soluble peptide (Figure 7). Using UV absorbance and UVRR spectroscopies we calculate that the NDQ20 supernatant contains ~0.076 mg/ml of peptide. The supernatant peptide concentration calculations are discussed in detail below.

Figure 7: Absorption spectrum of NDQ20 supernatant.

Soluble NDQ20 Supernatant Fraction Contains a PPII-like Peptide Conformation We used UVRR to examine the structure of the peptide in the NDQ20 supernatant (Figure 1e). The UVRR spectrum of the NDQ20 supernatant is similar to that of DQ20 (Figure 8). We 25 ACS Paragon Plus Environment


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observe AmIII3S bands at ~1250 cm-1, ~1215 cm-1, and ~1275 cm-1, which derive from Ψ angle distributions centered at ~150°, ~10°, and ~175° respectively (Figure 3b). As discussed above for DQ20, these Ψ angles are characteristic of PPII-like, turn-like, and 2.51-helix-like secondary structures, respectively. From the Ψ angle distributions we conclude that the peptide in the NDQ20 supernatant has the same secondary structure as DQ20. However, the widths of the PPIIlike and 2.51-helix-like Ψ angle distributions (and AmIII3S bands) of the NDQ20 supernatant are larger than that of DQ20 (Figure 3). This indicates that the PPII-like structure of the NDQ20 supernatant is less ordered compared to that of DQ20. Also, we find that the AmIP and AmIS bands of the NDQ20 supernatant are found at ~1680 cm-1, which is similar to that of DQ20 and indicates Gln side chains that are hydrogen bonded to water.


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Figure 8: UVRR spectra of (a) TFA and (b) NDQ20 supernatant with (blue) and without (red) subtraction of TFA. (c) UVRR spectral comparison of (red) DQ20 and (blue) NDQ20 supernatant with TFA subtracted.



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TFA Contamination in NDQ20 Supernatant In the NDQ20 supernatant UVRR spectra, we observe a strong, dominant peak at ~1435 cm-1 and a weaker peak at ~1205 cm-1 that results from trifluoroacetic acid (Figure 8a and b). These bands derive from CO stretching and asymmetric C-F stretching bands of the trifluoroacetate ion. Thermo Fisher Scientific synthesized our Q20 using an Fmoc solid-phase peptide synthesis where TFA was used for peptide cleavage and as a cosolvent for HPLC purification (see Methods Section). As a result, TFA is a common impurity in our Q20 peptides.42 When NDQ20 is centrifuged most of it pellets out. However, because of TFA’s miscibility with water it remains in the supernatant.


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Figure 9: UVRR spectra (204 nm) of 10% (v/v) TFA in water at (a) pH=~-1.5, (b) pH=~+0.5, and (c) pH=~+12. At pH=~+12 TFA is deprotonated and at pH=~-1.5 TFA is predominantly protonated.

Figure 9b shows the 204 nm UVRR spectrum of 10% (v/v) TFA (pKa=~+0.5)43 in water at pH=~+0.5. This spectrum is consistent with that previously reported for deprotonated TFA 29 ACS Paragon Plus Environment


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(trifluoroacetate).44,45 Also, the Figure 9b UVRR spectrum is identical to that of deprotonated TFA (Figure 9c) indicating that the trifluoroacetate ion is selectively resonance enhanced at 204 nm. In contrast, the UVRR spectrum of protonated TFA (Figure 9a) significantly differs from that of trifluoroacetate and has a much smaller 204 nm UVRR crosssection. We assign the spectrum of trifluoroacetate based on the assignments of Robinson and Taylor44 and Klemperer and Pimentel.45 The most intense peak in the TFA UVRR spectrum is located at ~1435 cm-1 and is assigned to symmetric carboxylate stretching motion. We also observe peaks at ~1205 cm-1 and 1620 cm-1 that were previously assigned to asymmetric C-F stretching44 and asymmetric carboxylate stretching,44,45 respectively. We assign our UVRR spectrum of protonated TFA based on the assignments of Fuson et al.46 For protonated TFA, the most intense UVRR peaks are found at ~1795 cm-1 and ~1772 cm-1. We assign these bands to C=O stretching of TFA based on the work by Fuson et al.46 We also observe a band at ~1452 cm-1 that is assigned to C=O deformation.46 The bands at ~1014 cm-1 and 1177 cm-1 and ~1273 cm-1 are assigned to OH out-of-plane deformation, C-F stretching, and OH in-plane deformations, respectively, of protonated TFA.46 We found that the NDQ20 supernatant has a pH of ~+3.5. Therefore TFA is predominantly deprotonated in the NDQ20 supernatant. The peaks at ~1435 cm-1 and ~1205 cm-1 in the NDQ20 supernatant are assigned to the strongly resonance enhanced carboxylate stretching and the C-F stretching bands of the trifluoroacetate ion. Subtraction of the TFA UVRR spectrum from that of the NDQ20 supernatant reduces the intensity of the ~1435 cm-1 and ~1205 cm-1 bands with little effect on the other UVRR bands, including the structurally sensitive AmIII3S and AmI bands (Figure 8a).


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Concentration of TFA in the NDQ20 Supernatant The NDQ20 supernatant contains both TFA and the Q20 peptide. We determined the concentration of TFA in the NDQ20 supernatant from its UVRR spectrum. To do this we first calculated the Raman cross section of the ~1435 cm-1 band of trifluoroacetate using the following equation: 𝜎𝑖 =

𝐼𝑖 𝑘𝑟 𝐶𝑟 𝜎𝑟 𝐼𝑟 𝑘𝑖 𝐶𝑖

𝐴 +𝐴

(𝐴 𝑖 +𝐴𝑒𝑥 ) 𝑟

𝑒𝑥

eq. 2

where σi is the Raman cross section of the trifluoroacetate ~1435 cm-1 band, σr is the Raman cross section of an internal standard Raman band, Ii is the intensity of the ~1435 cm-1 trifluoroacetate band, and Ir is the intensity of the internal standard Raman band. The factors kr and ki are the spectrometer efficiencies for the Raman bands of trifluoroacetate and the internal standard, respectively, and Cr and Ci are the concentrations of trifluoroacetate and the internal standard, respectively. The term in parentheses approximately corrects for sample self-absorption where Aex is the sample absorbance at the excitation frequency, Ai is the sample absorbance at the trifluoroacetate Raman band of interest, and Ar is the sample absorbance at the internal standard Raman band. To determine the 204 nm UVRR cross section for the ~1435 cm-1 band of trifluoroacetate, we measured the UVRR spectrum of TFA at pH=~+12 using sodium perchlorate (NaClO4) as an internal Raman cross section standard. Raman cross section of NaClO4 was estimated to be ~1.18x10-27 cm2 molecule-1 sr-1 for 204 nm excitation by extrapolating the Raman cross section measurements of Dudik et al.47 From eq. 2 we calculate that the 204 nm Raman cross section of the ~1435 cm-1 trifluoroacetate band is ~1.08(±0.01)x10-26 cm2 molecule-1 sr-1. 31 ACS Paragon Plus Environment


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Using the Raman cross section of trifluoroacetate we can determine the concentration of TFA in the NDQ20 supernatant. Rearrangement of eq. 2 gives: 𝐶𝑖 =

𝐼𝑖 𝑘𝑟 𝐶𝑟 𝜎𝑟 𝐼𝑟 𝑘𝑖 𝜎𝑖

𝐴 +𝐴

(𝐴 𝑖 +𝐴𝑒𝑥 ) 𝑟

𝑒𝑥

eq. 3

We collected UVRR spectra of the NDQ20 supernatant using sodium perchlorate as an internal standard. The NDQ20 supernatant has a pH of ~+3.5; thus, essentially all of the TFA in the supernatant will be deprotonated and contribute to the ~1435 cm-1 band of trifluoroacetate. Because we know the 204 nm Raman cross section of the ~1435 cm-1 trifluoroacetate band, we can calculate the concentration of TFA in the NDQ20 supernatant using eq. 3. We find that the concentration of TFA in the NDQ20 supernatant is ~810±70 μM. Concentration of Peptide in the NDQ20 Supernatant Using the concentration of TFA in the supernatant, calculated above, we determined the absorbance due to TFA at 214 nm in the NDQ20 supernatant. We constructed a calibration curve for TFA in the NDQ20 supernatant at 214 nm using a standard addition method (Figure 10a). From the calibration curve we calculate that 810 μM TFA should result in an absorbance of ~0.0006 at 214 nm. For the NDQ10 supernatant we find an absorbance of ~0.0113±0.002 at 214 nm (Figure 7). Thus, the absorbance of the peptide in the NDQ20 supernatant is ~0.0107. We constructed a calibration curve using highly soluble DQ20 peptide to determine the concentration of peptide in the supernatant (Figure 10b). At 214 nm, DQ20 has a molar absorptivity of ~19220 M-1 cm-1. From the DQ20 calibration curve we calculate that the NDQ20 supernatant contains ~0.076 mg/mL (~24.8 μM) of the Q20 peptide.


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Figure 10: Absorbance calibration curve at 214 nm for (a) TFA and (b) DQ20.

TFA used in Solid-Phase Peptide Synthesis may Induce PPII-Like Conformation Our results show that the peptide in the NDQ20 supernatant has a PPII-like structure similar to that of disaggregated peptides and contains TFA. This is consistent with, Thakur and coworkers48,49 who showed that TFA alone causes disaggregation of polyQ peptides. Further, disaggregated polyQ peptides have previously been found to occur in PPII-like conformations.11 Thus, we hypothesize that the PPII-like structure observed in the NDQ20 supernatant results from the use of TFA during peptide synthesis. However, the mechanism by which TFA induces structural change in polyQ peptides remains poorly understood.



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Structure of SPPS Q20 and NDQ20 UVRR Characterization and Ψ Angle Distribution of SPPS Q20 The UVRR spectrum of SPPS Q20 is similar to that of NDQ1011, which was found to have a collapsed β-strand-like conformation (Figure 11).11 The SPPS Q20 UVRR spectrum shows an AmIII3S frequency of ~1245 cm-1, which is 5 cm-1 upshifted compared to that of NDQ10 (~1240 cm-1). This ~5 cm-1 AmIII3S frequency difference between SPPS Q20 and NDQ10 likely results from differences in peptide backbone hydration. Mikhonin et al. previously correlated the AmIII3S frequency to the Ψ angle for peptides in a variety of backbone solvation states.22 They found that the frequency of the AmIII3S band is sensitive to water solvation of the peptide backbone. Comparison of the AmIII3S frequency–Ψ angle correlations for crystalline peptides and solvated peptides shows an ~4 cm-1 upshift of the AmIII3S band in peptide crystals compared to peptides in aqueous solution. This is similar to the ~5 cm-1 upshift of the AmIII3S band we observe for SPPS Q20 compared to aqueous NDQ10. Thus, the AmIII3S frequency differences between NDQ10 and SPPS Q20 are most likely to result from water exposure of the peptide backbone. In agreement, the AmIP band of SPPS Q20 indicates that the side chain amide groups are not hydrogen bonded to water (see below) while NDQ10 side chain and backbone carbonyl groups are hydrogen bonded to water.11 See below for a detailed discussion of the SPPS Q20 AmI bands.


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Figure 11: UVRR spectra of (red) NDQ10 and (blue) SPPS Q20. The NDQ10 spectrum was adapted with permission from Punihaole, D. et al. (2017). J. Phys. Chem. B, 121(24), 5953-5967. Copyright 2017 American Chemical Society.

Punihaole et al.11 previously calculated the Ψ angle distribution of the NDQ10 peptide from the AmIII3S band using an equation derived by Mikhonin et al.22 for peptides in aqueous solution with an unknown hydrogen bonding environment (equation S4). Using this equation, Punihaole et al. calculated that aqueous NDQ10 has a Ψ angle distribution centered at ~140°, characteristic of a β-strand-like secondary structures.11 Here, we use the AmIII3S band of SPPS Q20 to calculate its Ψ angle distribution. However, because the AmIII3S frequency depends on both the Ψ angle and hydrogen bonding to the amide C=O and N-H groups,22,33 we must account for the AmIII3S frequency shift resulting from differences in hydrogen bonding between dissolved NDQ10 and SPPS Q20. Therefore, instead of using eq S4 for aqueous peptides, we used the measured AmIII3S frequency-ψ angle correlation for crystalline peptides derived by Mikhonin et al.22 (equation S5). This equation best models the hydrogen bonding and solvation environments expected for SPPS Q20. Using this equation we calculate that SPPS Q20 has a Ψ angle distribution centered at ~138°, which is characteristic of β-strand-like structures (Figure 12b) such as that observed for NDQ10 (Ψ = 35 ACS Paragon Plus Environment


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~140°).11 Also, it is important to note that the AmIII3S band and Ψ angle distribution of SPPS Q20 is significantly broader than that of NDQ10. This indicates that the β-strand-like conformation of SPPS Q20 is less ordered and contains more conformational variations compared to that of NDQ10.


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Figure 12: Ψ angle distribution of (a) NDQ10 previously calculated by Punihaole et al.,11 (b) SPPS Q20, and (c) NDQ20. The NDQ10 Ψ distribution was adapted with permission from



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Punihaole, D. et al. (2017). J. Phys. Chem. B, 121(24), 5953-5967. Copyright 2017 American Chemical Society.

UVRR Characterization and Ψ Angle Distribution of NDQ20 We investigated the structure of NDQ20 (Figure 1d) by measuring its UVRR spectrum (Figure 13). We find that the UVRR spectrum of NDQ20 in water is very similar to that of SPPS Q20 (Figure 12b) with an AmIII3S band at ~1246 cm-1. Using eq S5, we calculate that the NDQ20 Ψ angle distribution is centered at ~138°, which is characteristic of a β-strand-like conformation (Figure 12c).

Figure 13: UVRR spectra of (blue) SPPS Q20 and (red) NDQ20 in water.

Side Chain and Backbone Hydrogen Bonding in SPPS Q20 and NDQ20


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At 204 nm excitation, SPPS Q20 and NDQ20 both contain broad (~70 cm-1 FWHH) AmI bands centered at ~1670 cm-1 (Figure 14). In contrast to SPPS Q20, NDQ20 also contains a narrow AmI peak at ~1660 cm-1, which indicates strong peptide-peptide hydrogen bonding.

Figure 14: UVRR AmI spectral region of (a) NDQ20 and (b) SPPS Q20. The spectra were modeled as a sum of Gaussian and Lorentzian bands, shown in black, as described in the supporting information. The AmI bands are labeled in each spectrum.

We examined the 197-204 nm difference spectra of SPPS Q20 and NDQ20 to highlight the contributions of the AmIP bands (Figure 15a and c). We find that SPPS Q20 and NDQ20 both 39 ACS Paragon Plus Environment


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contain narrow AmIP bands located at ~1666 cm-1 and ~1662 cm-1 respectively. These frequencies are indicative of strong side chain-side chain and/or side chain-backbone hydrogen bonding such as that found in polyQ fibrils.17 Using eq. 1 we estimate that the Gln side chain ΔHint = ~-5.3 kcal/mol for SPPS Q20 and ~-5.6 kcal/mol for NDQ20.

Figure 15: 197-204nm UVRR difference spectra of (a) NDQ20 and (c) SPPS Q20, and 204-(197204)nm difference spectra of (b) NDQ20 and (d) SPPS Q20.

The increased relative intensity of the ~1662 cm-1 AmIP band in the UVRR spectra of NDQ20, compared to that of SPPS Q20, indicates that NDQ20 contains a larger population of Gln side chains involved in strong side chain-peptide hydrogen bonding. Also, the lack of an ~1680 cm-1


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AmIP band in NDQ20 and SPPS Q20 indicates that neither species contains a significant population of Gln side chains hydrogen bonded to water. From the 204-(197-204) nm spectra (Figure 15b and d), we find that both NDQ20 and SPPS Q20 have an AmIS frequency of ~1675 cm-1. This frequency is upshifted from that of polyQ fibrils (~1660 cm-1) indicating that NDQ20 and SPPS Q20 contain weaker backbone-peptide hydrogen bonding. Also, the AmIS bands of NDQ20 and SPPS Q20 are broad (~70 cm-1 FWHH) suggesting that the peptide backbone amides are found in a variety of hydrogen bonding environments that inhomogenously broaden the AmIS band.

Stability and Activation Barrier between the PPII-like and β-Strand-like Structures of PolyQ Punihaole et al.11 previously showed that the PPII-like and β-strand like conformations do not interconvert because of a high activation barrier between them. We find that the PPII-like polyQ structure of DQ1011 and DQ20 contains negligible peptide-peptide hydrogen bonding and extensive peptide-water hydrogen bonding. PPII conformations are thought to be stabilized by peptide backbone-water hydrogen bonding50,51 and/or the low-energy water-water hydrogen bonding structure of the PPII peptide solvation shell. 50,52–57 Thus, it is likely that the polyQ PPIIlike structure is also stabilized by these interactions. A PPII-like → β-strand-like polyQ structural transition would have to overcome the energy barrier(s) associated with breaking peptide backbone-water hydrogen bonds and/or disrupting the water-water hydrogen bonding of the PPII solvation shell. In contrast, the β-strand-like structure of NDQ1011 and NDQ20 contains significant side chainpeptide hydrogen bonding. β-strand structures are stabilized by peptide-peptide hydrogen 41 ACS Paragon Plus Environment


bonding between neighboring β-strands.

58

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Thus, polyQ β-strand conformations are likely

stabilized by their side chain-peptide hydrogen bonding. A β-strand-like → PPII-like polyQ structural transition would have to overcome the energy barrier associated with breaking peptidepeptide hydrogen bonds.

NDQ20 Fibrillization To determine if NDQ20 (Figure 1d) will from fibrils, we examined the UVRR spectrum of aqueous NDQ20 after incubation for ~5 hours at room temperature (~18°C) and low pH (pH = ~+2-3) (Figure 1f). Incubation of NDQ20 resulted in a UVRR spectrum similar to that of NDQ10 fibrils reported by Punihaole et al. (Figure 16b).17 After incubation, the AmIII3S band downshifts to ~1233 cm-1, which is characteristic of the antiparallel-β-sheet structure of polyQ fibrils (ψ=~148°) (Figure 17b) that contain dry fibril cores.17 We also observe a low intensity AmIII3S band at ~1210 cm-1 that corresponds to a Ψ distribution centered at ~123°. As discussed previously by Punihaole et al., Ψ angles of ~123° arise from sub-populations of parallel β-sheet in polyQ fibrils.17 The widths of both the parallel and antiparallel β-sheet Ψ angle distributions are larger for NDQ20 fibrils compared to NDQ10 fibrils. This indicates that the secondary structure of the NDQ20 fibrils is less ordered compared to NDQ10 fibrils.


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Figure 16: (a) UVRR spectra of NDQ20 (blue) and NDQ20 fibrils (red). (b) Comparison of the UVRR spectra of NDQ20 fibrils (red) and NDQ10 fibrils (blue) previously collected by Punihaole et al.17 The NDQ10 fibril spectrum was adapted with permission from Punihaole, D. et al. (2016). J. Phys. Chem. B, 120(12), 3012-3026. Copyright 2016 American Chemical Society.



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Figure 17: Ψ angle distribution of (a) NDQ10 fibrils and (b) NDQ20 fibrils. Both NDQ10 fibrils and NDQ20 fibrils contain ψ angle distributions that peak at ~148° and ~123°, which are characteristic of antiparallel β-sheet and parallel β-sheet conformations respectively.

Additionally, the AmI band downshifts to ~1660 cm-1 and increases in relative intensity as a result of formation of strong peptide-peptide hydrogen bonds, as previously observed in Q10 fibril formation.17 To determine the frequency of the AmIP and AmIS bands, we examined the 44 ACS Paragon Plus Environment

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197-204nm and 204-(197-204) nm UVRR spectra of NDQ20 after incubation (Figure 18a and b). We find that the AmIP band is located at ~1665 cm-1, which corresponds to a side chain ΔHint of ~-5.4 kcal/mol. This is similar to that previously observed for Q10 fibrils. 17,30 Also, we find that the AmIS band is located at ~1665 cm-1, which suggests strong backbone-backbone hydrogen bonding, as previously observed in polyQ fibrils.17

Figure 18: (a) 197-204 nm and (b) 204-(197-204) nm difference spectra of NDQ20 after ~5 hours of incubation.



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To confirm formation of fibrils, we collected TEM images of NDQ20 after incubation for ~1-2 days at room temperature and low pH (pH = ~+2-3). Figure 19 shows TEM images of NDQ20 fibrils and NDQ10 fibrils. The TEM images of NDQ10 fibrils were previously collected by Punihaole et al.17 We find that NDQ20 contains amyloid-like fibril aggregates with a similar morphology to that observed for NDQ10.17 These images confirm that NDQ20 forms fibrils when incubated at room temperature (~18°C) and low pH (pH = ~+2-3).


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Figure 19: TEM images of (a) NDQ10 fibrils previously collected by Punihaole et al.17 and (b) NDQ20 fibrils (Figure 1f). The NDQ10 fibril TEM image was adapted with permission from Punihaole, D. et al. (2016). J. Phys. Chem. B, 120(12), 3012-3026. Copyright 2016 American Chemical Society.

Comparison to Other Results Our insights into the structures of SPPS Q20 and NDQ20 generally agree with results recently published by Burra and Thakur.59 They investigated the structures of the insoluble polyQ peptide K2Q9PGQ4AQ4PGQ9PGQ9K2 (PGQ9A) in the solid-phase synthesis lyophilized powder (SPPS PGQ9A) and non-disaggregated (NDPGQ9A) forms using FTIR and CD spectroscopies. Burra and Thakur59 found that SPPS PGQ9A contains predominantly antiparallel β-sheet structure with minority populations of random coil and turn conformations. They conclude that SPPS PGQ9A must have weaker peptide-peptide hydrogen bonding compared to β-sheet fibrils because turn and random coil conformations have a less optimal hydrogen bonding pattern.59 This is in agreement with our UVRR data showing that SSPS Q20 is in a β-strand-like conformation with weaker hydrogen bonding compared to fibrils. In contrast to SPPS PGQ9A, Burra and Thakur showed that NDPGQ9A contains only antiparallel β-sheet conformation.59 Since random coil and turn conformations were not observed in NDPGQ9A, they concluded that NDPGQ9A must contain stronger peptide-peptide hydrogen bonding compared to SPPS PGQ9A.59 Their conclusions agree with our results showing that NDQ20 contains a β-strand-like structure and stronger peptide-peptide hydrogen bonding compared to SPPS Q20. 47 ACS Paragon Plus Environment


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Additionally, Burra and Thakur do not observe signs of NDPGQ9A fibrillization after incubation in water for 3 hours.59 In contrast, we find that NDQ20 forms fibrils after ~5 hours of incubation in water. It is possible that Burra and Thakur did not observe fibrillization of NDPGQ9A because it requires an incubation time longer than 3 hours to form fibrils. The work by Burra and Thakur59 qualitatively examined the secondary structures of SPPS PGQ9A and NDPGQ9A. From the secondary structures, they predicted the hydrogen bonding in each species. Our measurements quantitatively extend the work of Burra and Thakur59 by quantifying the peptide Ψ angle distribution and by directly measuring the peptide backbone and Gln side chain hydrogen bonding interactions for SPPS Q20 and NDQ20. As described above, our measurements provide new insights into the specific secondary structures and hydrogen bonding interactions experienced by insoluble polyQ species.

Implication of NDQ20 Fibrillization on the PolyQ Fibrillization Mechanism Currently, there are two major models for the polyQ fibrillization mechanism. The first model, proposed by Wetzel and coworkers6,60, argues that polyQ aggregation occurs via nucleated growth. In this mechanism, the nucleus is thought to be a thermodynamically unfavorable conformation of the peptide monomer, and fibrils elongate by the recruitment of monomeric units to the growing fibril.6,61 The second model for the polyQ aggregation mechanism, developed by Pappu and coworkers, proposes that polyQ peptides form inter- and intra-peptide hydrogen bonds that lead to the formation of non-fibrillar polyQ aggregates.12–14 They propose that these aggregates can undergo a conformational change to form fibrils.62 48 ACS Paragon Plus Environment

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Here, we show that the insoluble NDQ20 (Figure 1d) peptide will convert from non-fibril aggregates to fibrils in water. This result suggests that non-fibril polyQ aggregates in water can undergo a conformational transition into fibrils.

Conclusion The results of this work are summarized in table 4. Table 4: Summary of secondary structure and hydrogen bonding for forms of Q20 examined in this study AmIP υ (cm-1) DQ20

~1681

Side Chain HBonding S.C.-W.

DQ20 Fibrils

~1662

S.C.-P.

NDQ20 ~1662 S.C.-P. NDQ20 ~1680 S.C.-W. Supernatant SPPS Q20 ~1666 S.C.-P. NDQ20 ~1665 S.C.-P. fibrils S.C., side chain; W., Water; P., Sheet, antiparallel β-Sheet

AmIS υ (cm-1) ~1675

Backbone HBonding B.B.-W.

Ψ Angle (°)

Secondary structure

~10, ~175, ~150 ~145, ~123

Turn, 2.51-Helix, PPII ~1663 B.B.-P. P-β-Sheet, A-β-Sheet ~1672 B.B.-P. ~138 β-Strand ~1680 B.B.-W. ~10, ~175, Turn, 2.51-Helix, ~150 PPII ~1672 B.B.-P. ~138 β-Strand ~1665 B.B.-P. ~148, ~123 P-β-Sheet, A-β-Sheet Peptide; B.B., Backbone; P-β-Sheet, parallel β-Sheet; A-β-

We used UVRR spectroscopy to investigate the structures of Q20. NDQ20 is essentially insoluble in water (Figure 1d). In contrast, we find that disaggregation of Q20 renders the peptide (DQ20) highly soluble (Figure 1i). Using UVRR spectroscopy we find that DQ20 is in a PPII-like conformation with backbone and side chain amide groups hydrogen bonded to water. 49 ACS Paragon Plus Environment


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To examine the solubility of NDQ20 (Figure 1d), we collected UV absorption spectra of the NDQ20 supernatant (Figure 1e) after ultracentrifugation. We find that the supernatant has weak absorbance. Using UV absorbance and UVRR we find a concentration of ~0.076 mg/mL for the peptide in the NDQ20 supernatant. Using UVRR spectroscopy we find that the soluble fraction of NDQ20 is in a PPII-like conformation (Figure 1e) with Gln side chains and peptide backbone hydrogen bonded to water, similar to that of DQ1011 and DQ20. The presence of PPII-like peptide structure in the NDQ20 supernatant could result from the use of TFA in the peptide synthesis, which can disaggregate polyQ in the absence of HFIP.48,49 We used UVRR spectroscopy to investigate the structures of SPPS Q20 (Figure 1c) and NDQ20 (Figure 1d). We find that SPPS Q20 and NDQ20 are in β-strand-like conformations (ψ=~138°) similar to that previously observed for NDQ10.11 Using UVRR spectroscopy, we also probed the backbone and side chain amide hydrogen bonding interactions in SPPS Q20 and NDQ20. We find that both NDQ20 and SPPS Q20 contain backbone amides with weaker interand intra-peptide hydrogen bonding compared to that found in fibrils. In contrast the side chain amide groups in SPPS Q20 and NDQ20 are involved in strong side chain-peptide hydrogen bonding. The number of side chain-peptide hydrogen bonds in NDQ20 is greater than that of SPPS Q20. Upon incubation for ~5 hrs, we find that NDQ20 converts to fibrils (Figure 1f).17 These fibrils have a β-sheet secondary structure with side chain and backbone amides involved in strong peptide-peptide hydrogen bonding. This result, along with the observation that NDQ20 contains more side chain-peptide hydrogen bonds compared to SPPS Q20, suggests that high molecular weight, non-fibrillar polyQ aggregates can undergo a conformational transition into fibrils. The


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discovery that apparently insoluble polyQ peptides can form fibrils may allow for fibrillization studies on NDQ peptides in a β-strand-like conformation without the need for disaggregation.

Acknowledgements Funding for this work was provided by the University of Pittsburgh and the Defense Threat Reduction Agency (DTRA) HDTRA1-09-14-FRCWMD (RSJ, SEW, SAA). SEW gratefully acknowledges support through the Molecular Biophysics and Structural Biology NIH Training Grant (T32 GM 088119). Supporting Information Available. UVRR spectral fitting, Ψ angle distribution calculations, TFA absorption spectra, and a comparison between the UVRR spectra of DQ10 and DQ20 fibrils can be found in the supporting information. This information is available free of charge via the internet at http://pubs.acs.org.

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TOC Figure


UV Resonance Raman Structural Characterization of an (In)soluble

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